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Daratumumab är en antikropp som används vid behandling av multipelt myelom. Daratumumab är specifik för CD38, ytproteinet som uttrycks i stor mängd på myeloma plasmaceller. Vid antikroppsscreen orsakar daratumumab panreaktivitet då erytrocyter också uttrycker CD38. Dithiothreitol (DTT) behandling av testerytrocyter har visats kunna eliminera panreaktiviteten med daratumumab-antikropparna, detta genom att DTT klyver bort epitopet på CD38 som daratumumab reagerar mot utan att förstöra andra kliniskt relevanta antigen. På Södra Älvsborgs sjukhus skickas prover från daratumumab-behandlade patienter till Sahlgrenska sjukhuset för genotypning för att hitta kompatibelt blod. Förhoppningen är att den nya metoden ska möjliggöra antikroppsscreen på daratumumab-behandlade patienter vid Södra Älvsborgs sjukhus.Syftet med examensarbetet var att verifiera metoden för användning av DTT-behandlade testerytrocyter på plasma från daratumumab-behandlade patienter för att underlätta val av erytrocyter för blodtransfusion.Provmaterialet var venöst tagen patientplasma från 16 daratumumab-behandlade patienter testades mot 0,2 M DTT-behandlade- och obehandlade-testerytrocyter. DTT-behandlade testerytrocyter testades även mot kända antikroppar på plasmaprover från patienter som ej behandlats med daratumumab. Hållbarhetsstudie utfördes med behandlade BAS-testceller.Panreaktivitet sågs hos samtliga patientprover med obehandlade testerytrocyter. Vid test med DTT-behandlade testerytrocyter blev samtliga prover negativa. Behandlade testerytrocyter som testades mot kända antikroppar gav resultat som var oförändrat jämfört med originalscreen. Behandlade testceller var brukbara 25 dagar.DTT behandling av testerytrocyter är effektivt och billigt, resultatet var pålitligt då samtliga patientprover inte uppvisade panreaktivitet efter DTT-behandling av testerytrocyter. De DTT-behandlade erytrocyterna behöll kliniskt relevanta antigen efter behandling och var hållbara 25 dagar. Metoden anses som användbar för Södra Älvsborgs sjukhus. / Daratumumab is an antibody used for treatment of multiple myeloma. The antibody is specific for the surface protein CD38 which is being expressed in high quantity on myeloma plasma cells. Daratumumab is causing pan-reactivity during antibody screening due to regular erythrocytes also express CD38. Dithiothreitol (DTT) treatment of test erythrocytes has shown to eliminate the pan-reactivity caused by the daratumumab antibodies by cleaving the epitope on CD38 that daratumumab is specific to. Södra Älvsborgs hospital are currently sending patient samples from patients treated with daratumumab to Sahlgrenska hospital in Gothenburg for genotyping to find compatible blood.The purpose of the project was to verify the method for use of DTT-treated test erythrocytes on plasma from daratumumab treated patients to screen for antibodies and easier find compatible erythrocytes for blood transfusion at Södra Älvsborgs hospital.Plasma samples from 16 patients treated with daratumumab were tested with DTT-treated and untreated test erythrocytes. DTT-treated test erythrocytes were tested against samples with known antibodies from patients not treated with daratumumab. The cells durability was also tested.Pan-reactivity was shown with all daratumumab samples with non-treated test erythrocytes. Tests with DTT-treated test erythrocytes showed no pan-reactivity. Results from treated test erythrocytes tested against known antibodies were unchanged from original screening. The cells were durable for 25 days.DTT-treatment of test erythrocytes is effective and cheap, test results were reliable, all patient samples had their pan-reactivity eliminated. DTT-treated erythrocytes kept clinically significant antigens. The method is useful for clinical use at Södra Älvsborgs hospital.

Personer som lider av multipelt myelom (MM) behandlas ofta med daratumumab. Daratumumab fäster till CD38 på erytrocyter vilket leder till problem med förenlighetsprövning, såsom antikroppsidentifiering och screening. När daratumumab i patientens plasma binder till testerytrocyter, ger det upphov till panreaktivitet. Dithiothreitol (DTT), ett reduktionsmedel, denaturerar CD38 så daratumumabantikropparna inte kan binda in och panreaktiviteten motverkas. Genom att DTT-behandla testerytrocyter kan kliniskt viktiga antikroppar påvisas och säkrare blodtransfusioner genomföras. En nackdel med DTT är att även Kell-antigenet bryts ner och anti-Kell kan ej påvisas. Syftet med examensarbetet var att verifiera metoden för DTT-behandling av testerytrocyter utifrån Region Östergötlands metodbeskrivning. I studien ingick totalt 21 plasmaprover från totalt 10 daratumumabbehandlade patienter. Testerytrocyterna behandlades med DTT enligt hänvisande metodbeskrivning. Obehandlade samt DTT-behandlade testerytrocyter testades mot plasmaproverna utan tillsatta blodgruppsantikroppar. DTT-behandlade testerytrocyter testades därefter mot plasmaproverna med tillsatt känd antikropp. Resultatet visade på en daratumumabinterferens mot obehandlade testerytrocyter och en avlägsnad interferens mot DTT-behandlade testerytrocyter. Samtliga tillsatta antikroppar kunde identifieras förutom anti-K vilket var förväntat, samt anti-s. Anti-s är främst betydelsefull vid gravidimmunisering och är därför inte lika viktig i detta sammanhang. Positiva respektive negativa prediktiva värdet (PPV och NPV) utföll 100% respektive 50%. NPV blev sämre på grund av få sanna negativa resultat i studien. Resultatet för positive respektive negative percentage agreement (PPA och NPA) utföll 88,9% respektive 100%. Under studien var hemolys ett problem och vidare studier krävs för att undersöka hållbarheten på DTT-behandlade testerytrocyter. Slutsatsen är att metodbeskrivningen för DTT-behandling av testerytrocyter kan tillämpas med vetskapen att anti-K och anti-s ej kan detekteras. / People suffering from multiple myeloma are often treaded with daratumumab. Daratumumab binds to CD38 on red blood cells (RBC) which interferes with compatibility tests, such as antibody identification and screening. Dithiothreitol (DTT), a reducing agent, acts to denature CD38 so daratumumab cannot bind. DTT treatment of RBC reagents make it possible to detect clinically significant antibodies and safer transfusions can be performed. Anti-K could not be identified because DTT destroyed the antigen. The objective of this study was to verify the method of DTT treatment of RBC reagents based on the method description of the Östergötland region. Untreated and DTT treated RBC reagents, respectively, were tested against plasma samples containing no blood group antibodies. DTT-treated RBC reagents were then tested against the plasma samples with added known antibody. The results showed a daratumumab-interference against untreated RBC reagents and that the interference was eliminated with DTT-treated RBC reagents. All antibodies could be identified, except anti-K and anti-s. Anti-s is particularly important in pregnancy immunization and is therefore not as important in this context. Positive and negative predictive values (PPV and NPV) were 100% and 50% respectively. NPV was low due to few true negative results in this study. The result for positive and negative percentage agreement (PPA and NPA) was 88,9% and 100% respectively. During the study, hemolysis was a problem and further studies are needed to investigate the sustainability of RBCs. The conclusion is that the method description for DTT treatment of RBC reagents can be applied with the knowledge that anti-K and anti-s cannot be detected.

Thesis (Ph. D.)--University of Virginia, 2007. / Includes bibliographical references. Also available online through Digital Dissertations.

In this study the heat-induced gelation of two (readily available) proteins, which contain disulphide bonds, has been investigated over a range of protein concentrations in the presence and absence of the presence of the reductant, dithiothreitol at neutral pH. The proteins selected in this study were: β-Lactoglobulin and bovine serum albumin. These proteins have different number of disulphide bonds and possess different protein secondary structures. The influences of the reductant and protein concentration on their heat-induced gelation were explored to see whether the proteins were able to form protein hydrogels and that the mechanical properties of the resulting protein hydrogels were controllable. The tilting test tube method revealed that both proteins formed macroscopic hydrogels, at protein concentrations above the critical gelation concentration and that the critical gelation concentration was constantly lower in the presence of the reductant. Micro-DSC revealed that both proteins had completely denatured upon heating and that the denaturation temperature and enthalpy were significantly lower in the presence of the reductant. IR spectroscopy revealed that both proteins undergo major secondary structure transitions that resulted in the formation of fibers that are rich in β-sheet structure upon heating and that the protein lost some secondary structure before any heating and gained more β-sheet structure in the presence of the reductant. Both proteins had partially denatured before any heating in the presence of the reductant and that β-LG underwent aggregation that was accompanied by the loss of native β-sheet structure and the formation of intermolecular β-sheet structure, while that BSA underwent aggregation that was accompanied by the loss of native α-helix structure and the formation of intermolecular β-sheet structure. Cryo-TEM revealed that both proteins formed fibers (10 nm in diameter) that exist as single entities at low protein concentrations and become entangled into macroscopic networks, at protein concentrations above the critical gelation concentration and that more fibers and denser macroscopic networks were formed in the presence of the reductant. Oscillatory rheology revealed that both proteins formed macroscopic networks exhibit viscoelastic behaviour and that their elastic modulus had increased in the presence of the reductant and with increasing protein concentration.

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Andrea Kladivová Consultant: Václav Tvrdý, MSc. Supervisor: Assoc. Prof. Přemysl Mladěnka, PharmD., Ph.D. Title of diploma thesis: Chelation of copper ions by thiol containing chelators Copper is an essential trace element that is important for many physiological functions. On the other hand, disruption of copper homeostasis associated with its elevated level is dangerous for the organism, because of the formation of free radicals. Copper chelators can represent an effective tool in the treatment of such conditions. Due to the ability of a thiol group to bind metal ions and form a chelate, thiol containing chelators are promising compounds for the reduction of copper levels. Within this diploma thesis, four compounds containing a thiol group were tested for the ability to chelate copper ions. Spectrophotometric measurement was used for this determination. It is a simple, fast but precise method for determination of the chelation properties in vitro. In addition, the ability of these compounds to reduce cupric ions was examined. When using the basic hematoxylin method, all the tested substances proved they can chelate copper. Their efficacy was practically identical except for the...

Mechanistic aspects of carbonaceous aerosol toxicity were examined with respect to the ability of particles to catalyse reactive oxygen species-generating redox cycling reactions. To investigate the role of secondary organic material, we examined two systems. In the first, two-stroke engine exhaust particles were found to increase their ability to catalyse redox cycling in the presence of a reducing agent, dithiothreitol (DTT), when the exhaust was exposed to ozone. This occurred through deposition of redox-active secondary organic aerosol (SOA) onto the particle that was ten times more redox active per microgram than the primary engine particle. In the second system, naphthalene SOA formed highly redox active particles. Activity was strongly correlated to the amount of the 1,4- and 1,2-naphthoquinone measured in the particle phase. However, these species and the newly quantified naphthalene oxidation product 5-hydroxy-1,4-naphthoquinone accounted for only 30% of the observed DTT decay from the particles. Gas-particle partitioning coefficients suggest 1,4- and 1,2-naphthoquinone are not strong contributors to ambient particle redox activity at 25°C. However, a large number of redox active species are unidentified. Some of these may be highly oxidised products of sufficiently low vapour pressure to be atmospherically relevant. DTT activity of diesel particles was found to be high per unit mass. The activity was found to be associated with the insoluble fraction as filtration of the particles nearly eliminated DTT decay. Neither methanol nor dichloromethane extracts of diesel particles exhibited redox activity, indicating that the redox active species are associated with the black carbon portion of the particles. Examination of particle concentration techniques found that use of water condensation to grow and concentrate particles introduced a large organic artefact to the particles. Experiments with concentrated inorganic particles suggest that the source of this artefact is from irreversible uptake of water-soluble volatile organic compounds. Overall, carbonaceous redox active species can be thought of as a continuum from small, water-soluble species to redox active functionalities on elemental carbon backbones. In addition to clearly defined, quantifiable species, future research may need to consider examining broader chemical classes or redox-active chemical functionalities to overcome the inherent complexity of these constituents.

Mechanistic aspects of carbonaceous aerosol toxicity were examined with respect to the ability of particles to catalyse reactive oxygen species-generating redox cycling reactions. To investigate the role of secondary organic material, we examined two systems. In the first, two-stroke engine exhaust particles were found to increase their ability to catalyse redox cycling in the presence of a reducing agent, dithiothreitol (DTT), when the exhaust was exposed to ozone. This occurred through deposition of redox-active secondary organic aerosol (SOA) onto the particle that was ten times more redox active per microgram than the primary engine particle. In the second system, naphthalene SOA formed highly redox active particles. Activity was strongly correlated to the amount of the 1,4- and 1,2-naphthoquinone measured in the particle phase. However, these species and the newly quantified naphthalene oxidation product 5-hydroxy-1,4-naphthoquinone accounted for only 30% of the observed DTT decay from the particles. Gas-particle partitioning coefficients suggest 1,4- and 1,2-naphthoquinone are not strong contributors to ambient particle redox activity at 25°C. However, a large number of redox active species are unidentified. Some of these may be highly oxidised products of sufficiently low vapour pressure to be atmospherically relevant. DTT activity of diesel particles was found to be high per unit mass. The activity was found to be associated with the insoluble fraction as filtration of the particles nearly eliminated DTT decay. Neither methanol nor dichloromethane extracts of diesel particles exhibited redox activity, indicating that the redox active species are associated with the black carbon portion of the particles. Examination of particle concentration techniques found that use of water condensation to grow and concentrate particles introduced a large organic artefact to the particles. Experiments with concentrated inorganic particles suggest that the source of this artefact is from irreversible uptake of water-soluble volatile organic compounds. Overall, carbonaceous redox active species can be thought of as a continuum from small, water-soluble species to redox active functionalities on elemental carbon backbones. In addition to clearly defined, quantifiable species, future research may need to consider examining broader chemical classes or redox-active chemical functionalities to overcome the inherent complexity of these constituents.

Introduktion: Daratumumab är en monoklonal antikropp som används för att behandla patienter med multipelt myelom (MM), en typ av blodcancer som har sitt ursprung i B-lymfocyter. Daratumumab binder till CD38 som normalt uttrycks på erytrocyternas yta och ger upphov till panreaktivitet vid förenlighetsprövning, som kan maskera kliniskt signifikanta antikroppar. Ditiotreitol (DTT), ett reducerande lösningsmedel, denaturerar den extracellulära domänen av CD38 och därmed hämmar inbindning av daratumumab. DTT-behandling av testerytrocyter vid förenlighetsprövning möjliggör detektion av kliniskt relevanta alloantikroppar.  Syfte: Att verifiera en metod för DTT-behandling av erytrocyter vid förenlighetsprövning, för att detektera alloantikroppar av klinisk relevans och lättare få fram blod till patienter som medicineras med Darzalex.  Metod & materiel: I studien ingick två plasmaprover från patienter med daratumumab behandling. Testerytrocyterna behandlades med olika DTT mängd (80, 160 och 320 L). DTT-behandlade samt obehandlade testerytrocyter testades mot plasmaproverna, negativ kontroll (anti-K) och positiv kontroll (anti-C) vid olika dagar från tillredningsdatum.  Resultat: Daratumumab interferens mot obehandlade testerytrocyter observerades. Reaktionen för samtliga DTT-behandlade testceller vid varje analystillfälle blev negativt, oavsett DTT mängd. Resultaten för den negativa kontrollen visade att det slog positivt vid DTT mängden 80 samt 160 µL och negativ vid 320 µL. Alla reaktioner för den positiva kontrollen var 4+ vid samtliga DTT mängder oavsett analysdag.  Slutsats:  DTT-behandling av erytrocyter inaktiverar CD38 som motverkar panreaktivitet vid serologiska analyser, för att upptäcka eventuella kliniskt signifikanta antikroppar hos patienter som behandlas med daratumumab. Den optimala DTT mängden är 320 µL/1 ml 1% testerytrocyt med upp till 15 dagars hållbarhet vid kylförvaring. / Introduction: Daratumumab is a monoclonal antibody used to treat patients with multiple myeloma (MM), a type of blood cancer that originates in B lymphocytes. Daratumumab binds to CD38, which is expressed on the surface of erythrocytes. This leads to panagglutination in compatibility testing and can mask clinically significant antibodies. Dithiothreitol (DTT), a reductant, denatures the extracellular domain of CD38, thereby inhibiting daratumumab from binding. DTT treatment of RBC-reagents enables the detection of clinically relevant alloantibodies. Purpose: To verify a method for DTT treatment of erythrocytes in compatibility testing, in order to detect alloantibodies of clinical relevance and to ensure that transfusion recipient with Darzalex medication receive compatible blood transfusion. Method: Two plasma samples from daratumumab treated patients were included. RBC-regents were treated with different amounts of DTT (80, 160 and 320 μL). DTT-treated and untreated RBC-reagents were tested against plasma samples, negative control (anti-K) and positive control (anti-C) on different days.  Results: Daratumumab interference with untreated RBC-reagents were observed.  DTT treated RBC-reagents however, showed negative reaction regardless of the amount of DTT used. Results for the negative control showed positive reaction with 80 and 160 µL DTT and negative at 320 µL. Reactions for the positive control were all 4+ no matter the amount of DTT and day it was analyzed.   Conclusion: DTT treatment of erythrocytes inactivates CD38, which prevents panagglutination, to detect any clinically significant antibodies in patients with daratumumab treatment. The optimal DTT amount is 320 µL / 1 ml 1% RBC-reagents and a shelf life of up to 15 days when stored cold.